Responses In The Brain Of Mice Immunized With The Recombinant M3-M4 Loop Fragment Of Human NR1a Protein

The N-methyl-D-aspartate receptor (NMDAR) plays a pivotal role in the process of glutamate-induced excitotoxicity associated with many neurological disorders including stroke, epilepsy, traumatic brain injury and some neurodegenerative diseases, et al. Thus, a large variety of NMDAR antagonists have been evaluated for potential clinical use. However, most of these drugs produced either little benefit because of a narrow therapeutic time window for administration or significantly adverse effects at effective doses. Establishing a humoral autoimmune response targeting the NR1 subunit of NMDAR may be a novel therapeutic strategy. The previous studies led by our group demonstrated that the extracellular M3-M4 loop of NR1a which is the dominant subunit of human NMDAR might be more easily used as a molecular target in immunization intervention to control the activation of NMDAR. So, the vaccination strategy targeting the M3-M4 loop could be expected to build with therapeutic potential for excitotoxic brain injuries. To further verify the feasibility of this immunotherapeutic strategy, in this study, we used the prokaryoticly recombinant and expressed M3-M4 loop(163aa,18.8KD)to vaccinate mice by three routes(intranasal, subcutaneous and intraperitoneal). The histological and immuno- histochemical staining were applied to the mice brain sections in order to evaluate the safty of this immunization for the central nervous system(CNS). The results are listed below:1. Serum cytokine assay by ELISAVaccinated mice were sacrificed 1day after the second immunization and the sera were prepared for examination. In i.n. immunized mice, concentrations of IFN-γ, IL-4 and IL-6 increased (p<0.05), and in s.c. immunized mice, the level of TNF-α,IFN-γ and IL-4,IL-6 also increased significantly(p<0.01).2. Serum antigen-specific antibody IgG assay by ELISASera were prepared from mice 7 days after the last boosting. Serum antigen-specific IgG was negative in i.n. vaccinated mice. However, relatively high level of IgG antibodies (1:800) were induced by s.c. immunization and lower IgG antibody titers (1:400) were obtained in i.p. immunized mice. 3. Phenotypic analysis of T subsets in nasal-associated lymphoid tissue(NALT) and spleen by cytometrySingle-cell suspensions from NALT and spleen were prepared 7 days after the last boosting. In i.n. immunized mice, the percentages of CD4+ and CD8+ T cells in NALT and spleen increased(p<0.05, p<0.01). S.c. immunized mice had the increased percentages of CD4+ and CD8+ splenocytes and the CD4+/CD8+ ratio decreased(p<0.01).4. Histological and Immunohistochemical Studies The all mice brains were removed 7 days after the last boosting and the sections were prepared for HE, Nissl staining and immuno- histochemical staining. No pathological change was observed in various regions of the brains. c-Fos and activated microglia immunoreactivities were negative. In conclusion, our experiment demonstrated that the recombinant M3-M4 loop induced the specific immune responses in mice via subcutaneous and intraperitoneal routes and did not do damage to the brains, therefore thought to be safe for the CNS.